Intracellular bacterial toxins enter cells, modify targets, and in many cases ultimately destroy the targeted cells thereby contributing to the disease process. Currently, there are no techniques for blocking intracellular virulence factors once they have entered the cytosol of cells. Further, no techniques exist which utilize inactive mutants derived from a toxin in order to inhibit the wild-type toxin at the intracellular cite.
Clostridium difficile is the leading cause of hospital acquired diarrhea and pseudomembranous colitis, a multifactorial disease involving steps in colonization, adherence, inflammation and cellular intoxication. TcdA and TcdB are two large clostridial toxins (LCTs) produced by C. difficile and are involved in development of pseudomembranous colitis. TcdB, (SEQ ID NO: 1), the focus of this study, glucosylates isoforms of small GTPases Rho, Rac and Cdc42 within the effector binding region at residues Threonine-37 (Rho) or Threonine-35 (Rac and Cdc42). The physiological impact of TcdB's activity includes disruption of tight junctions, increased epithelial permeability, as well as actin condensation and cell death.
TcdB can be divided into enzymatic, translocation and receptor binding domains, although detailed analysis of these regions has not been carried out to date. The first 546 amino acids of TcdB contain the enzymatic region, which is followed by a putative translocation and receptor-binding domain. Enzymatic activity appears to require the amino-terminal 546 residues since amino or carboxy terminal deletions of this fragment decrease activity. Within the enzymatic region, tryptophan 102 has been shown to be essential for UDP-glucose binding. A conserved DXD motif within LCTs is essential for LCT glucosyltransferase activity. Other studies, involving analysis of chimeras of the TcdB and TcsL enzymatic domain suggest residues 364 to 516 confer substrate specificity.
Steps in cell entry by TcdB have been broadly defined, yet events subsequent to entry are not well understood. For example, while we have a profile of the time-course for TcdB cell entry, very little is known about post-entry events that lead to glucosylation. Steps between membrane translocation and substrate interaction are not understood in TcdB intoxication. In fact almost no information exists in this regard for any intracellular toxin. In the cytosol, TcdB is capable of glucosylating multiple substrates, but whether inactivation of Rho, Rac and Cdc42 in combination is necessary for complete intoxication, or if other substrates are targeted, is not known. It has been found that overexpression of Rho isoforms protects cells from TcdB, suggesting inactivation of all substrates may not be necessary for cellular intoxication. Interestingly, Rho has also been shown to regulate the suppression of apoptosis, so it is not entirely clear whether overexpression of Rho is protective at the substrate inactivation level or prevents events downstream of glucosylation. Additionally, while some TcdB-intoxicating events, such as depolymerization of actin, can be attributed to inactivation of Rho, other processes like apoptosis may be linked to activities other than substrate inactivation. Given TcdB's large size (˜270 kD), and broad impact on cell physiology, it is possible the toxin may possess yet undefined activities in addition to glucosylation.
It would be desirable to have a vaccine or therapeutic composition for inhibiting or preventing action of the C. difficile TcdB toxin.